deletions | additions       diff --git a/Methods_Bill_Siena_this_is__.md b/Methods_Bill_Siena_this_is__.md  index 29d71dc..fa403a6 100644  --- a/Methods_Bill_Siena_this_is__.md  +++ b/Methods_Bill_Siena_this_is__.md 
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The BglB gene was codon-optimized for E. coli and cloned into a pET29b+ vector using Gibson assembly. Site-directed mutagenesis, utilizing the Kunkel method was used to generate mutations to BglB via the Transcriptic cloud laboratory platform.   BglB mutant plasmids were incubated on ice, allowing the plasmid to permeate the E. coli's surroundings. Heat shock at 42 degrees C and immediate incubation on ice result in E. Coli's uptake of exogenous genetic material. The addition of terrific broth and incubation at 37 degrees C for an hour assist in the recovery of E. Coli and the incorporation of the plasmid. The recovered cells were spread evenly on LB  agar plates with kanamycin resistance  and were incubated at 37 degrees C overnight. Multiple colonies on the agar plates should be visible within 12 hours. Growth media, containing terrific broth and kanamycin solution, was created. A single colony was scraped from an the incubated LB  agar plate plates  and dipped into the growth media in a15 mL  falcon tube. The single colony contains the mutant plasmid, which contains a gene resistant to kanamycin. In the growth media, the transforming plasmid survives while the other bacteria are susceptible to this antibiotic. This process ensures that onlythe  transformed cell is cells are  able to grow. The15 mL  falcon tubes were tube was  covered and incubated  with shaking for 37 degrees C for 24 hours.  The previously incubated falcon  tube seals was centrifuged and the resulting supernatant was dumped, leaving only the growth pellet of the transformed cell. Induction media, containing terrific broth, kanamycin solution, and isopropyl β-D-1-thiogalactopyranoside (IPTG), was created, and resuspended the growth pellet. IPTG induces the expression of the target protein, by mimicking the inducer allolactose in the lac operon pathway. The falcon tube, containing the resuspended growth pellet and the induction media, were covered  and incubated with shaking for 37 degrees C. C for 24 hours.  [Purification]